chapter 5 result and discussion 5 result and discussion...
TRANSCRIPT
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 123
5 RESULT AND DISCUSSION
5.1 Identification and Authentification of Plants
Bhunimbadi churna consists of ingredients viz., Swertia chirata (whole plant),
Holarrhena antidysenterica (seed), Zingiber officinale (rhizome), Piper nigrum
(fruit), Piper longum (fruit), Cyperus rotundus (rhizome), Picrorrhiza kurroa
(rhizome), Plumbago zeylanica (root), and Holarrhena antidysenterica (stem bark),
These plants were identified by organoleptic and morphologic charcters and were
authenticated by Dr. M. S. Jangid, Professor, Department of Botany, Sir P. T. Science
College, Modasa. Photographes of plants used in churna are given in Fig 5.1and
herbarium sheet of plants are given in Fig 5.2.
1) Chirayata 2) Indraju 3) Sunth
(Swertia chirata) (Holarrhena antidysenterica) (Zingiber officinale)
4) Mari 5) Pippali 6) Nagarmoth
(Piper nigrum) (Piper longum) (Cyperus rotundus)
7) Katuki 8) Chitrak 9) Kada chhal
(Picrorrhiza kurroa) (Plumbago zeylanic) (Holarrhena antidysenterica)
Fig 5.1: Photograph of plants used in Bhunimbadi churna
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 124
Fig 5.2(A): Herbarium sheet of Swertia chirata (Whole plant)
Fig 5.2(B): Herbarium sheet of Holarrhena antidysenterica (Seed)
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 125
Fig 5.2(C): Herbarium sheet of Zingiber officinale (Rhizome)
Fig 5.2(D): Herbarium sheet of Piper nigrum (Fruit)
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 126
Fig 5.2(E): Herbarium sheet of Piper longum (Fruit)
Fig 5.2(F): Herbarium sheet of Cyperus rotundus (Rhizome)
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 127
Fig 5.2(G): Herbarium sheet of Picrorrhiza kurroa (Rhizome)
Fig 5.2(H): Herbarium sheet of Plumbago zeylanic (Root)
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 128
Fig 5.2(I): Herbarium sheet of Holarrhena antidysenterica (Stem bark)
5.2 Evaluation of Quality Control Parameters for Raw Material
5.2.1 Organoleptic parameters
Organoleptic characters like colour, odour and taste of all ingredients used in powder
form in Bhunimbadi churna were carried out. Organoleptic characters like colour,
odour and taste of all ingredients used in powder form in Bhunimbadi churna is
shown in Table 5.1
Table 5.1: Organoleptic characters of Bhunimbadi churna ingredients
Sr
no.
Name of the
ingredients Part used
Description
Colour Odour Taste
1 Chiryata Whole plant Yellowish green Pleasant Extremely
Bitter
2 Indraju Seed Brownish yellow Characteristic Bitter
3 Sunth Rhizome Yellowish white Aromatic Pungent
4 Mari Fruit Greyish black Aromatic Pungent
5 Pippali Fruit Deep green Aromatic Pungent
6 Nagarmoth Rhizome Brown Pleasant Bitter
7 Katuki Rhizome Dark brown Pleasant Bitter
8 Chitrak Root Greyish yellow Characteristic Bitter
9 Kada chhal Stem Bark Light yellow Characteristic Bitter
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 129
Photograph of powders of all ingredients of churna is shown in Fig 5.3.
1) Chirayata 2) Indraju 3) Sunth
(Swertia chirata) (Holarrhena antidysenterica) (Zingiber officinale)
4) Mari 5) Pippali 6) Nagarmoth
(Piper nigrum) (Piper longum ) (Cyperus rotundus)
7) Katuki 8) Chitrak 9) Kada chhal
(Picrorrhiza kurroa) (Plumbago zeylanic) (Holarrhena antidysenterica)
Fig 5.3: Organoleptic characteristics of ingredients used in Bhunimbadi churna
The powder of chiryata was yellowish green, indraju was brownish yellow, sunth was
yellowish white, mari was greyish black, pippali was deep green, nagermoth was
brown, katuki was dark brown, chitrak was grayish yellow and kada chhal was light
yellow. Chiryata, nagarmoth and katuki was pleasant; sunth, mari and pippali was
aromatic and indraju, chitrak and kada chhal was with characteristic odour. Indraju,
nagarmoth, katuki, chitrak and kada chhal was bitter; sunth, mari and pippali powder
was pungent taste and chiryata was extremely bitter.
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 130
5.2.2 Powder microscopic characteristics
Ayurvedic pharmacopoeia committee constituted by the department of AYUSH has
introduced certain quality parameters for the plants that are used in Indian system of
medicine. The determination of the correct identity of the genuine source of the plant
material required to be used in various preparations is one of the most important
criteria for the assurance of quality and reliability. Microscopic evaluation of
powdered drugs is the easiest and cheapest method for detecting the correct
authenticity of them. Powder microscopy of all ingredient of Bhunimbadi churna was
carried out.
5.2.2.1 Swertia chirata (whole plant)
Powder microscopy of S. chirata whole plant was carried out and was observed under
normal and polarized light for calcium oxalate crystals and the photograph of the
same given in the fig 5.4
Parenchymataous cells Fibers
Vessels Calcium oxalate crystal
Fig 5.4: Powder characteristics of S. chirata (Whole plant)
Microscopic study of S. chirata whole plant powder showed flattened and tangentially
elongated parenchymatous cells; simple pits fibers; and simple and bordered pitted
vessels. Abundance, minute acicular calcium oxalate crystals were found in secondary
cortex and phloem region.
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 131
5.2.2.2 Holarrhena antidysenterica (Seed)
Powder microscopy of H. antidysenterica seed was carried out and observed under
normal and polarized light for calcium oxalate crystals. The photograph of the same
given in the fig 5.5
Endosperm Oil globules
Crystals of calcium oxalate (prismatic & rosette)
Fig 5.5: Powder characteristics of H. antidysenterica (Seed)
Microscopy of H. antidysenterica seed showed light yellowish-brown fragments of
endosperm, oil globules, prismatic and rosette crystals of calcium oxalate.
5.2.2.3 Zingiber officinale (Rhizome)
Microscopy Z. officinale rhizome powder was carried out and was observed under
normal light and the photograph of the same given in the fig 5.6
Cortex cells Vessels
Starch grains Fibres
Fig 5.6: Powder characteristics of Z. officinale (Rhizome)
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 132
Microscopic study of rhizome of Z. officinale shown parenchymatous thin-walled
cortex cells, reticulate or spiral vessels, septate fibers and flattened, rectangular and
ovate starch grains.
5.2.2.4 Piper nigrum (Fruit)
Microscopy P. nigrum fruit powder was carried out and was observed under normal
light and the photograph of the same is given in the fig 5.7
Epicarp Stone cells
Perisperum
Fig 5.7: Powder characteristics of P. nigrum (Fruit)
Microscopical study of P. nigrum fruit powder showed, single layered epicarp;
slightly sinuous, tabular cells forming epidermis; endocarp composed of slightly
elongated and beaker-shaped stone cells; perisperm with parenchymatous cells having
a few oil globules and packed, abundant, oval to round, simple and compound starch
grains having few aleurone grains and oil globules.
5.2.2.5 Piper longum (Fruit)
Microscopy P. longum fruit powder was carried out and was observed under normal
light and the photograph of the same is given in the fig 5.8
Microscopic study of P. longum fruit powder showed deep moss green fragments of
parenchyma, oval to elongated stone cells, vessels, oil globules and round to oval,
starch grains.
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 133
Fragments of parenchym Stone cells
Vessels Oil globules and starch grains
Fig 5.8: Powder characteristics of P. longum (Fruit)
5.2.2.6 Cyperus rotundus (Rhizome)
Microscopy C. rotundus rhizome powder was carried out and was observed under
normal light and the photograph of the same is given in the fig 5.9
Epidermis cells Vessels
Fibers
Fig 5.9: Powder characteristics of C. rotundus (Rhizome)
Microscopic study of C. rotundus rhizome powder showed epidermis with circular to
oval, thin-walled, parenchymatous cells with small intercellular spaces, narrow,
simple, reticulate and scalariform thickening and oblique pore vessels and fiber like
closely packed sclerified cells.
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 134
5.2.2.7 Picrorrhiza kurroa (Rhizome)
Microscopy of P. kurroa powder was carried out and observed under normal light
and the photograph of the same is given in the fig 5.10
Cork cells Parenchyma cells
Vessels Fibres
Fig 5.10: Powder characteristics of P. kurroa (Rhizome)
Microscopic study of rhizome of P. kurroa powder showed dusty grey, groups of
fragments of tangentially elongated suberised cork cells, thick-walled parenchyma,
pitted vessels and aseptate fibers.
5.2.2.8 Plumbago zeylanica (Root)
Microscopy P. zeylanica root powder was carried out and was observed under normal
light and the photograph of the same is given in the figure 5.11.
Cork cells Trachieds Fibers
Starch grains within cells
Fig 5.11: Powder characteristics of P. zeylanica (Root)
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 135
Microscopic study of rhizome of P. Zeylanica root powder revealed dark brown,
cubical to rectangular cork cells, pointed ends and narrow lumen trachieds, large
polygonal to tangentially elongated parenchymatous cells varying in size and shape,
containing starch grains and some cells with yellow contents and fibers scattered
singly or in groups.
5.2.2.9 Holarrhena antidysenterica (Stem bark)
Microscopy H. antidysenterica stem bark powder was carried out and was observed
under normal and polarized light for calcium oxalate crystals and the photograph of
the same is given in the fig 5.12.
Cork cells Fiber Calcium oxalate prism
Stone cells
Fig 5.12: Powder characteristics of H. antidysenterica (Stem bark)
Microscopic study of rhizome of H. antidysenterica stem bark powder showed
tangentially elongated cork cells, non-lignified pericyclic fibres, rectangular to oval
stone cells and numerous pits often containing prismatic crystals of calcium oxalate.
5.2.3 Physical parameters of raw material
Physicochemical parameters like moisture content and pH, water and alcohol soluble
extractive and ash value of raw material of all ingredient of Bhunimbadi churna was
carried out. All observation were taken thrice (n=3) and values are given as Mean ±
SEM.
A. Moisture content and pH: Moisture contents and pH of all the ingredients of
churna were carried out and results are given in the Table 5.2.
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 136
Table 5.2: Moisture content and pH of churna ingredients
Moisture content was found maximum in Katuki (9.34±0.04%w/w), while minimum
in Chiryata powder (6.11±0.02%w/w). Moisture content of all the ingredients were
less than 10%w/w, which help in free flowing property of churna and prevent
microbial or fungal growth. pH of all ingredients were between 5.82 to 7.30 suggested
palatable formulation. Results indicated that raw materials were reliable.
B. Water and alcohol soluble extractives: Water/alcohol extractive value and
API limits of extractive were given in the Table 5.3.
Table 5.3: Water and alcohol soluble extractives of churna ingredients.
Sr
no.
Ingredients
of churna
Water soluble Alcohol soluble
(%w/w) * API limit (%w/w) * API limit
1 Chiryata 14.58±0.87 > 10 11.03±0.15 > 10
2 Indraju 24.97±0.92 ---- 18.55±0.07 > 12
3 Sunth 15.02±0.84 > 10 3.48±0.19 > 3
4 Mari 9.76±0.97 > 6 7.88±0.08 > 6
5 Pippali 8.43±0.75 > 7 7.67±0.12 > 5
6 Nagarmoth 13.66±0.85 > 11 5.67±0.12 > 5
7 Katuki 22.71±0.94 > 20 16.19±0.14 > 10
8 Chitrak 15.45±0.98 > 12 12.84±0.05 > 12
9 Kada chhal 22.78±0.30 > 10 19.32±0.10 > 18
Water soluble extractive value was found maximum in indraju powder
(24.97±0.92%w/w), while minimum in pippali powder (8.43±0.75%w/w). Alcohol
Sr
no.
Ingredients
of churna
Moisture content
(%w/w)
PH
1 Chiryata 6.11±0.02 5.90±0.02
2 Indraju 9.10±0.01 6.35±0.01
3 Sunth 8.25±0.04 6.22±0.05
4 Mari 7.67±0.05 7.30±0.03
5 Pippali 6.56±0.02 6.40±0.02
6 Nagarmoth 8.97±0.01 5.88±0.01
7 Katuki 9.34±0.04 6.33±0.02
8 Chitrak 7.45±0.03 6.41±0.04
9 Kada chhal 6.97±0.04 5.82±0.02
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 137
soluble extractive were maximum in kada chhal powder (19.32±0.10%), while
minimum in sunth powder (3.48±0.19%). Water soluble extractives were higher than
alcohol soluble extractive in all ingredients of churna. Results were complies with
The Ayurvedic Pharmacopoeia of India, New Delhi.
C. Ash value
Total ash, acid insoluble ash and water soluble ash of all ingredients of churna were
carried out and results are express in Table 5.4.
Table 5.4: Different ash value of ingredients present in Bhunimbadi churna.
Sr
no.
Ingredients
of churna
Total ash Acid insoluble ash Water soluble
ash %w/w %w/w API Limit %w/w API Limit
1 Chiryata 2.75±0.02 < 6 0.74±0.03 < 1 1.20±0.01
2 Indraju 4.89±0.04 < 8 1.99±0.03 < 3 2.40±0.07
3 Sunth 3.97±0.01 < 6 1.02±0.04 < 1.5 1.40±0.02
4 Mari 4.18±0.02 < 5 0.22±0.07 < 0.5 0.52±0.03
5 Pippali 5.88±0.09 < 7 0.39±0.03 < 0.5 0.57±0.03
6 Nagarmoth 6.81±0.08 < 8 2.36±0.06 < 4 3.08±0.08
7 Katuki 4.51±0.02 < 7 0.35±0.05 < 1 0.49±0.09
8 Chitrak 2.43±0.04 < 3 0.48±0.02 < 1 0.67±0.02
9 Kada chhal 5.17±0.05 < 7 0.69±0.06 < 1 0.97±0.04
Total ash value, acid insoluble ash and water soluble ash were found maximum in
nagarmoth (6.81±0.08%w/w), (2.36±0.06%) and (3.08±0.08%) respectively. Total
ash value was found minimum in chitrak powder (2.43±0.04%w/w). Acid insoluble
ash and water soluble ash were minimum in mari powder (0.22±0.07%) and
(0.52±0.03%) respectively. Amount of inorganic material present in the plant is
represented as total ash, salt of sodium, potassium are soluble in water is represented
as water soluble ash, while silica like material remain insoluble in acid is represented
as acid insoluble ash. They are constant in plant and may help to diagnose the quality
of plant material. Results for all ingredients complies the limits of The Ayurvedic
pharmacopoeia of India indicating the material were of reliable quality.
5.3 Physical Parameters for Bhunimbadi churna.
Physical parameters like description, bulk density, tapped density, Hausner ratio,
Carr's index, angle of repose, pH, moisture content, water soluble, alcohol soluble,
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 138
total ash, water soluble ash, acid soluble ash of churna were carried out and results
are given in Table 5.5. All observation were taken thrice (n=3) and values are given as
Mean ± SEM.
Table 5.5: Physical parameters for Bhunimbadi churna.
Parameters Observations
Colors Grayish yellow
Odour Characteristic
Taste Bitter
Bulk density 0.3692g/cm3
Tapped density 0.5641g/cm3
Hausner ratio 1.528
Carr's index 34.55
Angle of repose 46.17º
pH 5.35±0.11
Moisture content 8.11±0.13%w/w
Extractive value
Water soluble
Alcohol soluble
Methanol soluble
Chloroform soluble
21.62±0.18%w/w
12.42±0.13%w/w
19.38±0.15%w/w
3.45±0.24%w/w
Ash value
Total ash 5.26±0.02%w/w
Water soluble ash 1.68±0.06%w/w
Acid insoluble ash 0.49±0.01%w/w
Results indicated that churna was greyish yellow, with characteristic odour and bitter
taste. Bulk density, tapped density, Hausner ratio and Carr's index indicated very poor
flow ability. Angle of repose indicated poor-must agitate, vibrate flow property. pH of
Churna were 5.35±0.11 and Moisture content of churna were 8.11±0.13%w/w. Water
soluble extractive 21.62±0.18%w/w was higher than others. Methanol soluble
extractive 19.38±0.15%w/w values was more than alcohol soluble extractive
12.42±0.13%w/w. Chloroform extractive 3.45±0.24%w/w values was the lowest than
others in Bhunimbadi churna. Total ash value was 5.26±0.02%w/w, water soluble ash
was 1.68±0.06%w/w and acid soluble ash was 0.49±0.01%w/w.
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 139
5.4 Qualitative Phytochemical Evaluation of Plant Material and Bhunimbadi
churna
All the ingredients were subjected for the various phytochemical tests carried out.
Results are given in Table 5.6
Table 5.6: Phytochemical tests for Bhunimbadi churna and ingredients
Sr
No.
Test
Ch
iryata
Ind
raju
Su
nth
Mari
Pip
ali
Narm
oth
Katu
ki
Ch
itra
k
Kad
a
chh
al
Bh
un
imba
di
chu
rna
1 Alkaloids
Dragandorff’s + + + + + + - - + +
Mayer’s - + + + + + - - + +
Wagner’s + + + + + + - - + +
2 Glycoside
General test + + + + + + + + + +
Foam test + + + - - + + + + +
3 Flavanoid
Shinoda test + + + - - + + + + +
Lead acetate test + + + + + - + + + +
4 Tannins
FeCl3 test + + + + + + + + + +
Gelatin test + + + - - - + + + +
5 Steroids and
Triterpenoids
Liberman burchard + + + + + + + + + +
Salkowaski’s + + + - - + + + + +
6 Phenolic
FeCl3 test + + + + + + + + + +
Acetic acid test + + + + + - + + + +
7 Carbohydrates
Molish test + + + + + + + + + +
Fehling’s test + + + + + + + + + +
8 Proteins and
Amino acids
Millon’s Test - - - - - - - - - -
Ninhydrin Test - - - - - - - - - -
Except katuki and chitrak all ingradients of churna had shown the presence of
alkaloid. All powders had shown the presence of glycoside, flavanoid, steroids &
triterpenoids, tannins and carbohydrate, while absence of protein and amino acid.
Bhunimbadi churna had shown the presence alkaloid, glycoside, flavanoid, steroids &
triterpenoids, tannins, carbohydrate while protein and amino acid were absent
indicating the presence of all ingredients in churna.
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 140
5.5 TLC of Raw Material and Churna with Laboratory Standard.
TLC provides a chromatographic finger print of drug. It is, therefore, suitable for
monitoring the identity and purity of drugs and for detecting adulteration and
substitution. TLC of all ingredient of churna and churna were carried out using silica
Gel 60F254 plate; samples in respective solvent, (alcohol extract of S. chirata , Z.
officinale and P. longum; methanol extract of P. nigrum, C. rotundus and P. kurroa;
chloroform extract of P. zeylanica and H. antidysenterica seeds and stem barks
methanol extract treated with 2ml 30% ammonium solution; respective solvent
system; derivatization by spraying reagents and heating the plate) for about 10min at
105°C. Results indicated all major spots corresponding to raw material as well as
churna are given in Figure 5.13 to 5.21.
Sample: Alcohol extract
Mobile phase: Toluene: Ethyl acetate:
Formic acid (5:4:1)
Spraying reagent: Anisaldehyde-
Sulphuric acid
Major spots at Rf: 0.25 (light violet),
0.40 (orange), 0.48 (violet), 0.68 (blue).
Fig 5.13: TLC of Swertia chirata
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 141
Sample: Methanol extract treated with
2ml 30% ammonium solution
Mobile phase: Toluene: Ethyl acetate:
Diethyl ether (70: 20: 10)
Spraying reagent: Dragendorff’s reagent
Major spots at Rf: 0.30, 0.45, 0.50 and
0.62 (all orange).
Fig 5.14: TLC of Holarrhena antidysentrica
Sample: Alcohol extract
Mobile phase: Toluene: Ethyl acetate
(93:7)
Spraying agent: Vanillin sulphuric acid
reagent
Major spots appear at Rf: 0.16, 0.22,
0.28(all violet) 0.50(red), 0.54(brown),
0.65, 0.68 and 0.78(light violet).
Fig 5.15: TLC of Zingiber officinale
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 142
Sample: Methanol extract
Mobile phase: Toluene: Ethyl acetate
(7:3)
Spraying reagent: Vanillin sulphuric
acid
Major spots at Rf: 0.20(light blue),
0.48(green) 0.50, 0.52 and 0.68(light
blue)
Fig 5.16: TLC of piper nigrum
Sample: Alcohol extract
Mobile phase: Toluene: Ethyl acetate
(90:10)
Spraying reagent: Vanillin Sulphuric
acid
Major spots at Rf: 0.28(green), 0.30,
0.42, 0.52 and 0.80(light brown).
Fig 5.17: TLC of piper longum
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 143
Sample: Methanol extract
Mobile phase: Toluene: Ethyl acetate
(9:1)
Spraying reagent: Vanillin sulphuric
acid
Major spots at Rf: 0.34(Brown),
0.74(light brown).
Fig 5.18: TLC of Cyperus rotundus
Sample: Methanol extract
Mobile phase: Chloroform: Methanol
(95:5)
Spraying reagent: Anisaldehyde
sulphuric acid
Major spots at Rf: 0.18(light blue),
0.48(blue) and 0.70(violet)
Fig 5.19: TLC of Picrorrhiza kurroa
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 144
Sample: Chloroform extract
Mobile phase: Toluene: Ethyl acetate
(3:1)
Spraying reagent: Anisaldehyde
sulphuric acid
Major spots at Rf: 0.20(light blue),
0.35(light green), 0.45(violet) and
0.84(yellow).
Fig 5.20: TLC of plumbago zeylanica
Sample: Methanol extract treated with
2ml 30% ammonium solution
Mobile phase: Toluene: Ethyl acetate:
Diethyl ether (70: 20: 10)
Spraying reagent: Dragendorff reagent
Major spots at Rf: 0.32, 0.45, 0.52 and
0.62 (all orange).
Fig 5.21: TLC of Holarrhena antidysenterica
The spots present in individual plant ingredient of churna were also present in TLC of
churna at same Rf and with identical colour, indicating incorporation of authentic
plant ingredient in churna.
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 145
5.6 HPTLC of Raw Material and Churna with Marker Compound.
CAMAG TLC scanner 3 and CAMAG Automatic TLC Sampler-4 spotting device
densitometry evaluation system with winCATS software was used for scanning of
thin layer chromatogram objects in reflectance or transmission mode by absorbance or
by fluorescence at 254 or 366nm or after derivatization, respectively. Rf value of
sample was evaluated using following formula
𝐑𝐟 =𝐃𝐢𝐬𝐭𝐚𝐧𝐜𝐞 𝐭𝐫𝐚𝐯𝐞𝐥𝐞𝐝 𝐛𝐲 𝐬𝐚𝐦𝐩𝐥𝐞 𝐟𝐫𝐨𝐦 𝐛𝐚𝐬𝐞𝐥𝐢𝐧𝐞
𝐃𝐢𝐬𝐭𝐚𝐧𝐜𝐞 𝐭𝐫𝐚𝐯𝐞𝐥𝐞𝐝 𝐛𝐲 𝐬𝐨𝐥𝐯𝐞𝐧𝐭 𝐟𝐫𝐨𝐦 𝐛𝐚𝐬𝐞𝐥𝐢𝐧𝐞
5.6.1 Estimation of mangiferin from Swertia chirata and churna by HPTLC
The standard solution of mangiferin 1mg/ml was prepared. Graded concentrations of
0.016, 0.027, 0.054, 0.080, and 0.107 µl volume of mangiferin were applied on a pre-
coated TLC silica gel 60 F254 plate (20cmx10cm). The concentration of the
mangiferin was 16, 27, 54, 80 and 100 ng/spot respectively. The mobile phase used
was ethyl acetate: methanol: water: formic acid (10:1:1:0.5). The photographs of
HPTLC plate of mangiferin, raw material of S. chirata and churna at 254nm and
366nm were shown in figure 5.22 and 5.23.
Track-1: 0.016 µg/µl of std mangiferin. Track-2: 0.027 µg/µl of std mangiferin.
Track-3: 0.054 µg/µl of std mangiferin. Track-4: 0.080 µg/µl of std mangiferin.
Track-5: 0.107 µg/µl of std mangiferin. Track-6: 5 µg/µl of swertia chirata
Track-7: 10 µg/µl of swertia chirata Track-8: 5 µg/µl of churna.
Track-9: 10 µg/µl of churna
Fig 5.22: Photograph of HPTLC plate mangiferin, S. chirata and churna at 254 nm
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 146
Fig 5.23: Photograph of HPTLC plate mangiferin, S. chirata and churna at 366 nm
HPTLC densitometric chromatogram of standard mangiferin, swertia chirata and
churna were shown in figure 5.24.
Fig 5.24: HPTLC densitogram mangiferin, S. chirata and churna
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 147
The peak areas of mangiferin for each concentration were recorded. Calibration curve
of mangiferin was prepared by plotting peak area against applied concentration of
mangiferin and shown in figure 5.25.
Fig 5.25: Calibration curve of mangiferin
A spot at Rf 0.39 was observed in the HPTLC chromatogram of the standard
mangiferin and raw material of S. chirata and churna. There was no interference with
other components present in the raw material and churna because they appear at
significantly different Rf values. The calibration curve of mangiferin was found to be
linear dependent on the concentration against peak area. The results of linearity
equation (y=10248x+1009) and correlation coefficient (R2=0.992) showed that within
the concentration range indicated, there was good correlation between peak area and
the corresponding concentration of mangiferin. The total concentration of mangiferin
in the raw material of S. chirata and churna were determined using calibration curve
of mangiferin. Raw material of S. chirata and churna were found to contain
mangiferin 0.21%w/w and 0.076%w/w respectively.
5.6.2 Estimation of 6-gingerol from Zingiber officinale and churna by HPTLC
The standard solution 1mg/ml 6-gingerol was prepared. Graded concentrations of
0.077, 0.128, 0.256, 0.384, and 0.512µl volume representing 77, 128, 256, 384 and
512ng/spot of 6-gingerol respectively were applied on a pre-coated TLC silica gel 60
F254 plate (20cmx10cm). The mobile phase was used n-hexane: ethyl acetate (50:50).
The photographs of HPTLC plate of 6-gingerol, raw material of Z. officinale and
churna at 254nm, 366nm and after derivatization were shown in figure 5.26.
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 148
At 254nm
At 366nm
After derivatization
Track-1: 0.077 µg/µl 6-gingerol. Track-2: 0.128 µg/µl 6-gingerol.
Track-3: 0.256 µg/µl 6-gingerol. Track-4: 0.384 µg/µl 6-gingerol.
Track-5: 0.512 µg/µl 6-gingerol. Track-6: 5 µg/µl Z.officinale
Track-7: 10 µg/µl of Z. officinale Track-8: 5 µg/µl churna.
Track-9: 10 µg/µl of Churna
Fig 5.26: HPTLC Photograph plate of 6-gingerol, Z. officinale and churna
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 149
The separated bands on the HPTLC plates were scanned over the wavelength of 400–
800 nm. The source of radiation utilized was tungsten lamp. The maximum
absorbance was found to be at 533 nm. HPTLC densitometric chromatogram of
standard 6-gingerol, zingiber officinale and churna are shown in figure 5.27.
Fig 5.27: HPTLC densitogram of 6-gingerol, Z. officinale and churna.
A spot of 6-gingerol was observed at Rf 0.60 in the HPTLC chromatogram of the
standard, raw material of Z. officinale and churna. There was no interference with
other components present in the raw material and churna because they appear at
significantly different Rf values. The peak areas of 6-gingerol for each concentration
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 150
were recorded. Calibration curve of 6-gingerol was prepared by plotting peak area
against applied concentration of 6-gingerol and is shown in figure 5.28.
Fig 5.28: Calibration curve of 6-gingerol
The calibration curve of 6-gingerol had given linearity equation (y=6191x+239) and
correlation coefficient (R2=0.993) indicating calibration curve was found to be linear
dependent on the concentration against peak area. The results of showed that within
the concentration range indicated, there was good correlation between peak area and
the corresponding concentration of 6-gingerol. The total concentration of 6-gingerol
in the raw material of Z. officinale and Bhunimbadi churna was determined using
calibration curve of 6-gingerol and were found to be 0.54%w/w and 0.06%w/w.
5.6.3 Estimation of piperin from Piper nigrum, Piper longum and churna by
HPTLC
The standard solution 1mg/ml piperin was prepared. Graded concentrations of 0.4,
0.8, 1.2, 1.6, 2.0 µl volume of Piperin indicating 400, 800, 1200, 1600 and 2000
ng/spot Piperin respectively were applied on a pre-coated TLC silica gel 60 F254
plate (20cm x 10cm). The mobile phase was used Toluene: Ethyl acetate: Formic acid
(3:1:0.1). The photographs of HPTLC plate showing Piperin in varying concentrations
and in raw material of p. nigrum, p. longum and churna at 254nm and 366nm are
shown in figure 5.29 and 5.30.
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 151
Fig 5.29: Photograph of HPTLC plate of piperin, P. nigrum, P. longum and
churna at 254 nm.
Track-1: 0.04 µg/µl Piperin. Track-2: 0.06 µg/µl Piperin.
Track-3: 0.08 µg/µl Piperin. Track-4: 0.10 µg/µl Piperin.
Track-5: 0.12 µg/µl Piperin. Track-6: 2 µg/µl of P. nigrum.
Track-7: 4 µg/µl P. nigrum. Track-8: 2 µg/µl P. longum.
Track-9: 4 µg/µl P. longum. Track-10: 2 µg/µl churna.
Track-11: 4 µg/µl churna.
Fig 5.30: Photograph of HPTLC plate of piperin, P. nigrum, P. longum and
churna at 366nm.
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 152
The separated bands on the HPTLC plates were scanned over the wavelength of 400–
600 nm. The source of radiation utilized was D2 lamp. The maximum absorbance was
found to be at 334 nm. HPTLC densitometric chromatogram of standard piperine, P.
nigrum, P. longum and churna were carried out and shown in figure 5.31.
Fig 5.31: HPTLC densitogram of piperin, P. nigrum, P. longum and churna.
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 153
A spot of Piperine was observed at Rf 0.28 in the HPTLC chromatogram of the
standard piperin and raw material of p. nigrum, p. longum and churna. There was no
interference with other components present in the raw material and churna because
they were separted at significantly different Rf values. The peak areas of Piperin for
each concentration were recorded. Calibration curve of piperin was prepared by
plotting peak area against applied concentration of piperin and shown in figure 5.32.
Fig 5.32: Calibration curve of piperin
The calibration curve of piperin was with linearity equation (y=11539x+136.4) and
correlation coefficient (R2=0.986) indicated the calibration curve of piperin was
linear and dependent on the concentration against peak area within the concentration
range indicated. There was good correlation between peak area and the corresponding
concentration of piperin. The total concentration of piperin in the raw material of P.
nigrum, P. longum and churna was determined using calibration curve of piperin and
were found to be 2.87%w/w,1.53%w/w and 0.24%w/w respectively.
5.6.4 Estimation of conessine in H. antidysenterica seed & stem bark and
churna by HPTLC
The standard solution 1mg/ml conessine was prepared. Graded concentrations of 0.2,
0.4, 0.6, 0.8, 1.0µl volume of conessine repesenting 200, 400, 600, 800 and 1000
ng/spot conessine respectively were applied on a pre-coated TLC silica gel 60 F254
plate (20cmx10cm). The mobile phase toluene: ethyl acetate: diethyl amine
(7.0:2.0:1) was used.
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 154
The photographs of HPTLC plate showing conessine in varying concentation and in
raw material of H. antidysenterica seed & stem bark and churna at 254nm, 366nm
and after derivatization are shown in figure 5.33.
At 254.
At 366.
After derivatization.
Track-1: 0.04 µg/µl conessine. Track-2: 0.06 µg/µl conessine Track-3: 0.08 µg/µl
conessine. Track-4: 0.10 µg/µl conessine. Track-5: 0.12 µg/µl conessine. Track-6: 2
µg/µl H.seed. Track-7: 4 µg/µl H. seed. Track-8: 2 µg/µl H. stem bark. Track-9: 4
µg/µl of H. stem bark. Track-10: 2 µg/µl churna.Track-11: 4 µg/µl churna.
Fig 5.33: HPTLC plate of conessine, H. antidysenterica seed & stem bark and churna
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 155
The separated bands showing conessine on the HPTLC plates were scanned over the
wavelength of 400–800 nm. Tungsten lamp was utilized as a source of radiation. The
maximum absorbance was found to be at 520nm. HPTLC densitogram of conessine in
varying amount and in raw material of H. antidysenterica seed & stem bark and
churna were carried out and is shown in figure 5.34.
Fig 5.34: HPTLC densitogram conessine, raw material of H. antidysenterica seed,
stem bark and churna
A spot of conessine was observed at Rf 0.86 in HPTLC chromatogram of the standard
conessine, raw material of H. antidysenterica seed, stem bark and churna. There was
no interference with other components present in the raw material and churna because
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 156
they appear at significantly different Rf values. The peak areas of conessine for each
concentration were recorded. Calibration curve of conessine was prepared by plotting
peak area against applied concentration of conessine and shown in figure 5.35.
Fig 5.35: Calibration curve of conessine
The calibration curve of conessine was found with linearity equation (y=280x+1653)
and correlation coefficient (R2=0.995) indicated calibration curve of conessine was
linear and dependent on the concentration against peak area. There was good
correlation between peak area and the corresponding concentration of conessine. The
total concentration of conessine in the raw material of H. antidysenterica seed, stem
bark and churna was determined using calibration curve of conessine and were found
to be 0.19%w/w,0.90%w/w and 0.083%w/w.
HPTLC methods developed for estimation mangiferine, 6-gingerol, piperin and
conessine in individual plant and combination as churna was accurate, linear, rugged,
simple and rapid. Total concentration of mangiferine, 6-gingerol, piperin and
conessine estimated was well complies with the reference standard. This method can
be utilize the estimation of plant containing mangiferine, 6-gingerol, piperin and
conessine individual and in combination. Result showed that HPTLC technique can
be successfully used for estimation of mangiferine, 6-gingerol, piperin and conessine
in Bhunimbadi churna.
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 157
5.6.5 HPTLC finger printing of Bhunimadi churna
HPTLC finger printing of Bhunimadi churna was carried out using silica Gel 60F254
plate; churna in methanol extract using toluene: ethyl acetate: formic acid (5: 1.5: 0.5
v/v) solvent system. Plate was observed under UV-light at 254nm and 366nm.
Derivatization by anisaldehyde sulphuric acid spraying reagents and heating the plate
for about 10min at 105°C. Solvent system was modified by trial and error to give
separation of maximum compounds. Photographs of plate and chromatogram of
churna is given in Figure 5.36.
Fig 5.36: HPTLC fingerprinting of Bhunimbadi churna
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 158
Compounds separated in HPTLC fingerprinting were observed under UV light at
254nm and 366nm and after derivatization are tabulated with their Rf value in Table
5.7.
Table 5.7: Peaks of HPTLC of Bhunimbadi churna
Peak At 254 At 366 After Derivatization
Max Rf Max Rf Max Rf
1 0.12 0.25 0.05
2 0.25 0.37 0.09
3 0.33 0.44 0.19
4 0.45 0.56 0.25
5 0.56 0.62 0.45
6 0.59 0.73 0.56
7 0.62 0.83 0.62
8 0.73 0.88 0.68
9 0.75 0.73
10 0.82 0.82
11 0.85 0.95
12 0.87
Result indicated that HPTLC finger printing of Bhunimadi churna under UV light at
254 nm, had showed 13 blue colour spots at Rf 0.25, 0.33, 0.45, 0.56, 0.59, 0.62, 0.73,
0.75, 0.82, 0.85, 0.87, 0.95. UV light at 366nm had showed 8 spots at Rf 0.25, 0.37,
0.56, 0.62 were purple; 0.44, 0.56 were dark green and 0.73, 0.83, 0.88 were black.
After derivatization with the anisaldehyde-sulphuric acid reagent had showed 11 spots
at Rf 0.05, 0.68 were dark purple; 0.09 was yellow; 0.19 was light purple; 0.25 was
purple; 0.45 was green; 0.56 was dark green; 0.62 was purple; 0.73, 0.95 were black
and 0.82 was light pink.
Human fingerprint is unique and will not match with others. Similarly HPTLC
fingerprinting of plant and their formulation are also unique and not match with other
plants and other formulations. Hence HPTLC fingerprinting helps in identification,
establishing uniformity, detection of adulteration, addition and omission of ingredient
of formulation and determination of quality of raw material used in the formulation.
5.7 Heavy Metals Analysis:
Analysis of heavy metal in Bhunimbadi churna like Lead (Pb), Cadmium (Cd),
Arsenic (As), Mercury (Hg), were carried out using Labindia Inductively coupled
plasma-optical emission spectroscopy (ICP-OES). Results are shown in Table 5.8
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 159
Table 5.8: Heavy metal analysis
Sr No. Heavy metal Bhunimbadi churna (ppm) API limit (ppm)
1 Lead (Pb) 9.40 10
2 Cadmium (Cd) 0.3 0.3
3 Arsenic (As) 2.15 10
4 Mercury (Hg) Absent 1
Heavy metals after entering in the human body can’t be removed easily. On long
usage they accumulated in the body ultimately produce toxicity by hindering the
normal physiological activity. Hence WHO and various pharmacopoeias have
prescribed the limit for heavy metals. Results showed that concentration of lead and
cadmium in Bhunimbadi churna were 9.40ppm and 0.3ppm respectively and
concentration of arsenic was 2.15ppm and mercury was absent in Bhunimbadi churna.
The presence of heavy metal in Bhunimbadi churna were less than the limit
prescribed in The Ayurvedic Pharmacopoeia of India.
5.8 Microbial Analysis
Microbial analysis of Bhunimbadi churna included total viable aerobic count, total
yeast and mould, E. coli, S. Typhi count. The results are given in Table 5.9
Table 5.9: Microbial analysis
Sr No. Microbial analysis Bhunimbadi churna API Limit
1 Total aerobic viable count 410 cfu/gm 105 /gm
2 Total yeast and mould Absent 103 /gm
3 E. coli Absent Absent
4 Salmonella Sp Absent Absent
Pathogenic bacteria in vegetative and non-vegetative forms are harmful to human
body because they produce diseases. Hence WHO and pharmacopoeia of the advance
countries have prescribed the limit for total aerobic viable count, total yeast and
mould, E. coli and Salmonella species. Results indicated that Total aerobic viable
count was 410 CFU/gm and Total yeast and mould, E. coli and Salmonella species
were absent in Bhunimbadi churna. This indicated that total aerobic viable count, total
yeast and mould, E. coli and Salmonella Species in Bhunimbadi churna were lower
than the limit prescribed in The Ayurvedic Pharmacopoeia of India.
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 160
5.9 In-vitro Antioxidant Activity of Bhunimbadi churna.
In-Vitro the antioxidant activity of the methanol extract of the churna was carried out
by DPPH-Free radical scavenging activity, H2O2 scavenging activity and Ferric
reducing antioxidant power antioxidant models, using ascorbic acid as reference
standard.
5.9.1 DPPH-Free radical scavenging activity
The absorbance maximum of a stable DPPH radical in methanol was measured at
517nm. The decrease in absorbance of DPPH caused by antioxidants (ascorbic acid,
methanol extract of churna) was determined at 517nm. The reaction results in the
scavenging of free radical by hydrogen donation change the color from purple to
yellow. Results are illustrated in Figure 5.37. Values are express as means± SD of
triplicate analyses and Significance P<0.05 compared to control
Fig 5.37: DPPH radicals scavenging activity of methanol extract of churna
The results indicated that methanol extracts of churna and ascorbic acid had showed
significant dose dependents reduction in free radical to the corresponding hydrazine in
the antioxidant principles of DPPH. The odd electron of DPPH radical becomes
paired with hydrogen from a free radical scavenging antioxidant to form the reduced
DPPH-H. The resulting decolorization is stoichiometric with respect to number of
electrons captured. This indicated antioxidant activity of Bhunimbadi churna.
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 161
5.9.2 Hydrogen peroxide scavenging activity
Absorbance of H2O2 at 230nm was determined with different concentrations of
methanol extract of churna and standard ascorbic acid and also after 10min against a
blank solution containing phosphate buffer without H2O2. The percentage inhibition
activity was calculated. H2O2 scavenging activity was graphical presented by plotting
graph using concentration vs. absorbance and shown in Figure 5.38. All the tests were
performed in triplicate and the graph was plotted with the average of three
observations.
Fig 5.38: H2O2 scavenging activity of methanol extract of churna
Result demonstrated that methanol extract of churna and ascorbic acid had shown
significant concentration dependant scavenging of H2O2. H2O2 is an intermediate
during endogenous oxidative metabolism and mediates radical oxygen formation such
as OH, which may be used to predict the scavenging capability of antioxidants in
biological systems. H2O2 has only a weak activity to initiate lipid peroxidation, but its
activity as an active oxygen species comes from its potential to produce the highly
reactive hydroxyl radical through the Fenton reaction. Thus, removal of H2O2 is very
important for protection of food systems.
The antioxidant activity of churna was expressed as IC50 which is defined as the
concentration (μg/ml) of methanol extract of churna inhibits the formation of DPPH
radicals by 50%. IC50 in DPPH and H2O2 models was calculated by linear regression
and results are given in table 5.10.
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 162
Table 5.10: IC50 of methanol extract of churna in DPPH and H2O2 models.
Sr No. Antioxidant model IC50 (µg/ml)
Ascorbic acid Methanol extract of
Bhunimbadi churna
1 DPPH model 14.26 62.80
2 H2O2 model 35.64 75.45
The IC50 values in DPPH model were found to be 62.80 and 14.26 µg/ml for
methanol extracts of churna and ascorbic acid respectively. IC50 values in Hydrogen
Peroxide scavenging model were found to be 35.64 and 75.45 μg/ml for Ascorbic acid
and Methanol extract of churna respectivily. Methanol extract of churna had exibited
higher antioxidant activity in DPPH and H2O2 models.
5.9.3 Ferric reducing antioxidant power activity
Methanol extracts of churna was subjected to Ferric reducing power activity.
Reducing power acitivity was determind by increase in absorbances. Concentration
vs. absorbance of methanol extracts of churna and ascorbic acid were graphically
plotted and given in Fig 5.39. Values are means± SD of triplicate analyses.
Fig 5.39: Ferric reducing antioxidant activity of methanol churna
The results showed that methanol extract of churna and ascorbic acid had
demonstrated dose dependent antioxidant activity in ferric reducing power model.
Antioxidant activity of methanol extract of Bhunimbadi churna in DPPH, H2O2 and
ferric reducing antioxidant model may attributed due to presence of phytoconstituents
like; alkaloid, glycoside, flavanoid, phenolic compounds and tannins.
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 163
5.10 Antidiabetic Activity of Churna in Alloxan Induced Diabetic Rat Model.
Bhunimbadi churna, prepared as per ‘Bruhad Nighantu Ratnakar’ and was subjected
to assess its antidiabetic potential. Alloxan, when it is administered parenterally,
intravenously, intraperitoneally or subcutaneously, causes a massive reduction in
insulin release by the destruction of β-cells of the islets of Langerhans and induces
hyperglycaemia, hence Alloxan induced diabetics in rat model was selected to
establish the efficacy of churna.
5.10.1 Acute toxicity: Acute toxicity of Bhunimbadi churna was carried out in rat.
Four hrs. fasted animals were orally administered 100-2000mg/kg body weight
Bhunimbadi churna in 5% v/v Tween 80 solution in five animal with different dose.
Animals were observed individually during the first 30minute after dosing, every 4hr
during the first 12hr, and thereafter for 3 days for behaviour change. Neurological
change and autonomic change. Results are given in table 5.11.
Table 5.11: Acute toxicity of Bhunimbadi churna
Profile 30min 4hrs 8hrs 12hrs 3days
Behavioral Alertness NC NC NC NC NC
Restlessness NC NC NC NC NC
Irritability NC NC NC NC NC
Fearfulness NC NC NC NC NC
Neurological Spontaneous activity NC NC NC NC NC
Reactivity NC NC NC NC NC
Touches response NC NC NC NC NC
Pain response NC NC NC NC NC
Gait NC NC NC NC NC
Autonomic Defecation NC NC NC NC NC
Urination NC NC NC NC NC
Mortality Death NC NC NC NC NC
NC= No change
Acute toxicity study revealed the non-toxic nature of churna up to 2000mg/kg. All the
animals were alive, healthy and active during the observation period. No lethality or
single toxic reactions were found at any of the doses selected until the end of the
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 164
study period. Considering this, 200mg/kg and 400mg/kg doses were selected for
further study.
For antidiabetic study animals were divided into five groups. Normal control group
(Group I) received laboratory diet and water ad libitum. Rests of the rats were fasted
18hrs and were intraperitonialy given a single dose 100mg/kg, body weight Alloxan
monohydrate. Animals were maintained for two days in diabetic condition for well
establishment of diabetes. Diabetic control group (Group II) was fed with Standard
Laboratory Diet and water ad libitum. Reference standard group (Group III) was daily
treated orally with 5mg/kg body weight Glibenclamide in water for 14 days. Group IV
and Group V were daily treated with dose of 200 and 400mg/kg b.w Bhunimbadi
churna respectively for 14 days. Blood samples were collected from the retro-orbital
plexus of 8hr fasted and anesthetized animals by slight exposure to ether on first, 7th
and 14th
days and biochemical parameters like; body weight, blood glucose, serum
total cholesterol, serum triglycerides, serum HDL, serum VLDL, serum LDL, serum
total protein and albumin proteins, serum creatinine and urea were determined.
Results were statistically analysed using one-way analysis of variance (ANOVA)
followed by Dunnett’s test using GraphPad Instat3 software and expressed as Mean ±
SEM (n=6), Group II was compared with Group I, Group-III, IV and groupV.
**P<0.01, *P<0.05.
5.10.2 Body Weight: Body weight of animal in all the groups were checked at the
end of 1st, 7
th and 14
th days and results are given in Figure 5.40 and 5.41.
Fig 5.40: Antidiabetic activity of Bhunimbadi churna by rat body weight
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 165
Fig 5.41: Antidiabetic activity of Bhunimbadi churna by rat body weight
Result showed that after first, 7th
and 14th
days, body weight in normal control group,
reference standard group, group-IV and V had significantly increased (p<0.05). While
diabetic control group showed significantly (p<0.05) decreases in body weight. It was
concluded that induction of diabetes with Alloxan is associated with a characteristic
loss of body weight, which is due to increased muscle wasting and loss of tissue
proteins. Diabetic rats treated with the Bhunimbadi churna showed significant dose
dependent increase in body weight, which may be due to its effect in controlling
muscle wasting; by reversal or antagonizing. This was supported by the result in
reference standard group receiving Glibenclamide which significantly prevent the loss
of body weight in experimental diabetic rats.
5.10.3 Blood glucose level: Blood glucose level of animal in all the groups were
corded on first, 7th
and 14th
days and results are graphically represented, day wise in
Figure 5.42 and group wise in Figure 5.43.
Alloxan used as an agent for the development of experimental diabetes to induce
selective dysfunctioning of pancreatic β-cells. In-vitro studies have shown that
Alloxan is selectively toxic to pancreatic β -cells, leading to the induction of cell
necrosis. The cytotoxic action of alloxan is mediated by reactive oxygen species, with
a simultaneous massive increase in cytosolic calcium concentration, leading to a rapid
destruction of β-cells characterized by reduced circulating concentration of insulin;
poor insulin sensitivity or insulin resistance and poor glucose tolerance resulting in
high blood glucose level.
*
**** **
**
***
**
***
130
140
150
160
170
180
190
200
Group I Group II Group III Group IV Group v
gm
Treatment in Days
Body weight (gm)
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 166
Fig 5.42: Antidiabetic activity of Bhunimbadi churna by rat blood glucose level
Fig 5.43: Antidiabetic activity of Bhunimbadi churna by rat blood glucose level
Result showed that after first, 7th
and 14th
days, blood glucose level in diabetic control
group animals was significantly increased. While diabetic animals receiving
Bhunimbadi churna 200 and 400mg/kg of body weight and Gibenclamide
significantly decreased blood glucose level (p<0.05). Bhunimbadi churna in Alloxan
induced rats produced significant dose dependent decreased in blood glucose level
(p<0.05) indicating antidiabetic activity.
******
**
**
**
**
**
**
**
50
100
150
200
250
300
350
400
Group I Group II Group III Group IV Group v
mg/
dl
Treatment in Days
Blood glucose Level (mg/dl)
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 167
5.10.4 Lipid profile: Lipid profile of serum cholesterol level, serum triglyceride,
serum HDL, VLDL and LDL animal in all the groups was checked at the end 14th
days and results are graphically presented in Figure 5.44.
Fig 5.44: Antidiabetic activity of Bhunimbadi churna by rat lipid profile
Results indicated that after 14th
days serum cholesterol level significantly (p<0.05)
dose dependently decreased in diabetic animal treated with Bhunimbadi churna.
There was also significant (p<0.05), and dose dependent decreased in the serum
triglyceride level in diabetic animal receiving Bhunimbadi churna. There was
significant (p<0.05), dose dependent increased in the serum HDL level in diabetic
animal treated with Bhunimbadi churna. There was significant (p<0.05), dose
dependent decreased in the serum VLDL level and serum LDL level in diabetic
animal treated with Bhunimbadi churna. Gibenclamide had also significantly
normalized Alloxan induced diabetic animal by decreasing serum cholesterol, serum
triglyceride, serum VLDL and serum LDL and increasing serum HDL (p<0.05).
Results indicated the Bhunimbadi churna had shown antidiabetic activity by
significant (p<0.05) and dose dependent decreasesing the serum cholesterol, serum
triglyceride, serum VLDL, serum LDL and increasesing the serum HDL level in
alloxan induced diabetic rats.
The levels of serum lipids are usually elevated in diabetes mellitus and such an
elevation represents a risk factor for coronary heart disease. This abnormal high level
of serum lipids is mainly due to the uninhibited actions of lipolytic hormones on the
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 168
fat depots mainly due to the action of insulin. Under normal circumstances, insulin
activates the enzyme lipoprotein lipase, which hydrolyses triglycerides. However, in
diabetic state lipoprotein lipase is not activated due to insulin deficiency resulting in
hyper-triglyceridemia and insulin deficiency is also associated with hyper-
cholesterolemia due to metabolic abnormalities. Hence, the diabetic rats showed
hyper-cholesterolemia and hyper-triglyceridemia and the treatment with churna
significantly (p<0.05) and dose dependently lowered cholesterol and triglyceride
levels.
5.10.5 Serum urea: Serum urea level is one of the parameter of renal profile. Serum
urea level was checked in the animals of all the groups on 14th
days and results are
graphically presented in Figure 5.45.
Fig 5.45: Antidiabetic activity of Bhunimbadi churna by rat serum urea
Results indicated that there was a significant rise in serum urea level (p<0.05) in
Diabetic control rats. Bhunimbadi churna (200 and 400 mg/kg) and Glibenclamide
significantly reduced the elevated serum urea level (p<0.05).
5.10.6 Serum creatinine: Serum creatinine level is also one of the parameter for
renal profile. Serum creatinine level was measured in the animals of all the groups on
14th
days and results are graphically presented in Figure 5.46.
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 169
Fig 5.46: Antidiabetic activity of Bhunimbadi churna by rat serum creatinine
There was also significant rise in serum creatinine level in alloxan induced rats
(p<0.05). Bhunimbadi churna (200 and 400 mg/Kg) and Glibenclamide significantly
reduced the elevated serum creatinine level (p<0.05).
Serum urea and creatinine is metabolic end product of amino acid and protein, in the
blood that would be normally excreted in the urine by kidneys’ nephrons vessels.
When blood glucose levels are consistently too high affects the kidneys ability to
filter and filtration rate in glomeruls, cause End Stage Renal Disease (ESRD). Urea
formation is influenced by a number of factors such as liver function, protein intake
and rate of protein catabolism. Elevation of serum urea and creatinine level in alloxan
induced diabetic rat and significantly normalize by application of glibenclamide and
Bhunimbadi churna in Alloxan induced diabetic rats.
5.10.7 Serum albumin and total protein: Similar to serum urea and serum
creatinine, serum albumin and total protein are also the parameters for the renal
profile. Serum albumin and total protein level were measured in the animals of normal
control group, diabetic control group, standard reference group and Bhunimbadi
churna 200 and 400 mg/kg body weight treated group on 14th
days and results are
graphically presented in Figure 5.47.
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 170
Fig 5.47: Antidiabetic activity of Bhunimbadi churna by rat albumin and total
protein.
Results indicate that there was a significant decreased in serum albumin and total
protein level (p<0.05) in Diabetic control rats. Bhunimbadi churna (200 and 400
mg/kg) and Glibenclamide significantly elevated serum albumin and total protein
level (p<0.05).
Low levels of albumin (microalbuminuria) in the urine cause incipient nephropathy.
Microalbuminuria is a sensitive and early marker of diabetic nephropathy, other forms
of renal dysfunction, and endothelial permeability throughout the vascular tree. It
predicts progression of kidney disease leading to End Stage Renal Disease,
development of cardiovascular disease, retinopathy, peripheral vascular disease and
total mortality. Decreased in serum albumin and total protein level in Alloxan induced
diabetic rat and significantly normalize by administration of Glibenclamide and
Bhunimbadi churna in Alloxan induced diabetic rats.
Bhunimbadi churna improved renal profile in Alloxan induced diabetic rats by
decreasing serum urea and creatinine level and increasing serum albumin and total
protein indicating renal protecting activity of Bhunimbadi churna.
**
**
***
**
**
****
0
1
2
3
4
5
6
7
8
g/
dl
Serum albumine Serum Total Protein
Renal profile (gm/dl)
Chapter 5 RESULT AND DISCUSSION
Bhavik Ph. D Dissertation 171
Antidiabetic activity of Bhunimbadi churna was concluded by exhibiting regaining
body weight, lowering blood glucose level, improving lipid profile by decreasing
serum cholesterol, serum triglyceride, serum VLDL & serum LDL level and
increasing serum HDL level and improving renal profile by decreasing serum urea
and creatinine level and increasing serum albumin and total protein.
Bhunimbadi churna was shown the presence alkaloid, glycoside, flavanoid, steroids
and triterpenoids, tannins and carbohydrate which conformed by phytochemical
analysis. Antioxidant activity, antidiabetic activity, improvement in lipid profile and
renal profile exhibited by Bhunimbadi churna may be due to phytoconstituents
present like; alkaloid, glycoside, flavanoid, steroids and triterpenoids, tannins and
carbohydrate. Flavonoids, steroids/ triterpenoids, alkaloids and phenolics are known
to be bioactive antidiabetic principles. Flavonoids are known to regenerate the
damaged beta cells in Alloxan induced diabetic rats.
Present investigation justifies the traditional therapeutic claim as antidiabetic activity
of Bhunimbadi churna. However further phytochemical investigation may provide
isolation and identification of specific compound/s from Bhunimbadi churna which
may be responsible for antioxidant activity, antidiabetic activity and improving lipid
and renal profile.